L2 β-lactamase contributes to ceftolozane-tazobactam resistance in Pseudomonas aeruginosa

The prevalence of non-susceptibility to ceftolozane-tazobactam (C/T) among Pseudomonas aeruginosa remains low, but novel mechanisms of C/T resistance are of concern. Herein, we describe a novel P. aeruginosa genotype associated with high-level C/T resistance (>256/4 µg/mL) in a single patient. Whole-genome sequencing of the isolate was compared to that of a susceptible isolate cultured from the same patient 2 months earlier. Analysis of the sequences revealed two different P. aeruginosa high-risk clones: sequence type (ST)111 followed by ST235. The C/T-resistant ST235 isolate contained five copies of a genetic element composed of an L2 β-lactamase gene ( bla L2 ) and a truncated ampR L2 transcriptional regulator gene, which are commonly found together in Stenotrophomonas maltophilia strains and have not been reported to mediate resistance to C/T. Comparative genomic analysis with other P. aeruginosa isolates failed to identify alternative explanations for the observed C/T resistance. We found that exogenous expression of bla L2 increased C/T minimum inhibitory concentrations (MICs) in genetically distinct P. aeruginosa strains. A screen of our archived isolates identified two P. aeruginosa clinical isolates, PS2045 and PS2046, with one and two copies, respectively, of the genetic element containing bla L2 and truncated ampR L2 . Interestingly, disruption of the gene bla L2 but not the truncated ampR L2 in PS2045 led to a decrease in C/T MIC. Thus, we report a novel mechanism of C/T resistance in P. aeruginosa partially mediated by an L2 β-lactamase independently of its canonical regulator, AmpR L2 .